A method for the separation of peptides and α-amino acids (original) (raw)
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Journal of Chromatography A, 2009
This review discusses different liquid chromatographic and capillary electrochromatographic approaches to the separation and quantitation of peptides using silica-based and polymeric-based columns with emphasis on liquid chromatography. Mass spectrometry detection and quantitation of peptides using labeled and label-free procedures, will also be discussed, as well as the effect of amino acids' properties on the solubility of peptides, an important parameter that influences the selection of the mobile phase. A discussion of different column packing materials, reversed-phase, cyclodextrins, macrocyclic antibiotics, porous graphitic carbon, mixed-phases, and normal-phase will be included, as well as a short discussion of multi-dimensional approaches for the separation of complex peptide mixtures.
Analytical Biochemistry, 1975
Polyacrylamide gel electrophoresis of Fluorescamine-modified peptides is a rapid (90 mitt), sensitive (< 1 nmole), and reproducible method which may be used to resolve peptides without the limitations with respect to molecular size or water solubility found in other analytical systems. Fluorescamine-modified peptides, ranging in size from 3 to 215 amino acids, have been examined in a variety of buffer systems between pH 2.3 and 9.8 and gel concentrations varying between 4% and 16% acrylamide. Similar peptides were examined by high-voltage paper electrophoresis and detected by Fluorescamine. Peptides of low solubility have been examined in the presence of urea. The method reported here was used to resolve small peptides differing in size by a single amino acid, as well as peptides of the same size differing only by a single charge. It was successfully employed as a criterion for homogeneity of peptides in the course of purification for amino acid sequence analysis.
Separation of peptides by strong cation-exchange high-performance liquid chromatography
Journal of Chromatography A, 1985
The effects of pH and gradient conditions on the separation of a series of ten peptides (9-36 residues) and carboxamidomethylated troponin I (CM-TnI, 178 residues) on a new commercially available strong cation-exchange silica based 300-A column (Synchropak S300) were examined. The elution times of the peptides were linear with respect to their net charge at pH 3.0 and pH 6.5. The basic protein CM-TnI (pZ % 9.5) and peptides with net charges from + 2 to + 10 were separated with linear AB salt gradients varying from 5 mM to 10 mM B per min (A = 5 mM KH2P04 buffer, pH 6.5 or 3.0; B = 5 mM KH2P04 buffer, pH 6.5 or 3.0, containing 1 M KCl). All peptides and CM-TnI were eluted with KC1 concentrations below cu. 0.6 M. The advantage of performing cation-exchange chromatography over anionexchange chromatography was demonstrated for the separation of peptides which, while acidic or weakly basic at neutral pH, through protonation of the acidic functions results in positively charged peptides at pH 3.0. 0021-9673/85/%03.30 0 1985 Elsevier Science Publishers B.V.
A Method for the Sample Handling and Analysis of Bio-Active Peptides
1998
A method has been developed for the sample handling and analysis of bio-active peptides. Based on electrospray ionization mass spectrometry (ESI-MS), the method is applicable to the target analysis of known peptides. ESI-MS is used to determine the molecular mass of the peptide and provisional identification is based on matching the molecular mass with that of known peptides in a database. Disulfide bridge reductive alkylation is used to determine the number of cysteines in the peptide as well as the presence of disulfide bridges. The peptide is then enzymatically digested and the digestion products analyzed by ESI-MS. The resulting mass map is compared to the masses predicted from the structure of the peptide. Finally, the peptide and its enzymatic fragments are analyzed by liquid chromatography (LC) ESI-MS using collisional activated disassociation (CAD) conditions which promote the formation of product ions from which it is possible to determine the amino acid sequence of the peptide. The developed method was applied to the analysis and identification of a-conotoxin GI. The monoisotopic molecular mass was determined to be 1436.42 ± 0.13 u (n=6). A search of the in-house database determined that the only match within one mass unit of the calculated molecular mass was a-conotoxin GI with a theoretical molecular mass of 1436.48 u. The error between the calculated and theoretical values was 42 ppm. Reductive alkylation indicated the presence of four cysteines and two intramolecular disulfide bridges which was consistent with the structure of a-conotoxin GI. LC-ESI-MS analysis of the tryptic digestion products indicated the presence of two fragments with masses of 564.18 and 1122.45 u which were in agreement with the predicted products. Under CAD conditions, Y series ions were observed from which the entire sequence of the larger tryptic fragment was determined.
Separation and Analysis of Peptides and Proteins
Analytical Chemistry, 1995
Our studies of Levantine viper venom have demonstrated that the venom is a rich source of biomedically important proteins and peptides. The venom contains metalloproteases: thrombolytic, fibrin-degrading lebetase, an endothelial cell apoptosis inducing metalloprotease (VLAIP), factor X activator (VLFXA); serine proteases: factor V activator, bradykininreleasing serine proteases, β-fibrinogenase, α-fibrinogenase and chymotrypsin-like protease and different other enzymes such as phosphodiesterase, 5`-nucleotidase, ribonuclease, phospholipase A2s and L-amino acid oxidase. Among nonenzymatic components venom contains: nerve growth factor, vascular endothelial growth factor, disintegrins, C-type lectins.
Biochemical and Biophysical Research Communications, 1998
IUPAC-IUB JOINT CONNISSION ON BIOCHEMICAL NOMENCLATURE 22.4 Removal of residues 22.5 Substitution of side chains of residues 22.5.1 Acylation of a side-chain amino group 22.5.2 Other substituents named as prefixes 22.5.3 Acylation by a side-chain carboxyl group 22.6 Partial sequences (fragments) 22.7 Peptides with reversed sequence and enantiomers 22.8 Peptide analogues 22.9 Summary of modification nomenclature References Appendix Amino Acids with Trivial Names * Square brackets are required to indicate replacement, but are not used for most other modifications.
Review: Determination of Amino Acids by Different Methods
2021
The article review discusses a variety of methods for determining amino acid concentrations. The process used to assess the amino acid composition or content of proteins, peptides, and other pharmacological preparations is known as amino acid analysis. Proteins and peptides are macromolecules made up of linear polymers of covalently bound amino acid residues. The qualities of a protein or peptide are determined by the sequence of amino acids in the molecule. On the basis of recent publications, this review discusses recent methods developments in amino acids analytics. It aids in the updating and systematisation of knowledge in this field. The advancement of analytical methods is highlighted, along with the benefits and limitations of the numerous procedures used for the manufacture, separation, and determination of amino acids. The preparation requirements for methods analysis vary depending on the type of material. As a result, the focus of the review has been on detection and est...
Journal of Chromatography B: Biomedical Sciences and Applications, 1996
HPLC and CE have been applied to the separation of some newly synthesized substances, including nonapeptides from the intrachinary region of insulin, insulin-like growth factors I and II (IGF I and II) and some penta-and hexapeptides. All the peptides are satisfactorily separated using a reversed-phase HPLC system with a C~8 stationary phase and mobile phases of 20-40% acetonitrile (v/v) and 0.2% trifluoroacetic acid in water (v/v). The best CE separation of IGF I and II has been achieved in a 30 mM phosphate buffer (pH 4-5), whereas 150 mM phosphoric acid (pH 1.8) is optimal for the insulin nonapeptides. The latter electrolyte is also suitable for the CE separation of the hexapeptides, as is a micellar system containing 20 mM borate-50 mM sodium dodecyl sulfate (pH 9.0). Complete CE resolution of the D-and L-forms is possible in a 50 mM phosphate buffer (pH 2.5) containing 10 mM fl-cyclodextrin. UV spectrophotometric detection was used throughout, at wavelengths from 190 to 215 nm. The CE procedures are, in general, preferable to HPLC separations, as they exhibit better separation efficiencies, are faster and consume smaller amounts of analytes and reagents.
Peptide fractionation by acid pH SDS-free electrophoresis
ELECTROPHORESIS, 2011
SDS-free polyacrylamide gel electrophoresis is an effective alternative approach to peptide fractionation. Here we describe a discontinuous buffer system at acid pH that improves the separation of acidic peptides from tryptic digestion. MOPS and chloride act as trailing and leading ions, respectively, in this system, while histidine operates as counterion and buffers all solutions. In these electrophoretic conditions, peptides with pI below 5.5 migrate with low overall electrophoretic mobilities but high differences from one another, which allows for their efficient resolution. In silico analysis of several proteomes shows that the acid pH system allows a peptide simplification of 2.5-fold with respect to the total peptide mixture, and still a proteome coverage of about 95% is achievable. A straightforward method with a protocol including proteomic studies was achieved for SDS-PAGE of proteins, enzyme treatment and further peptide fractionation by SDS-free acid PAGE.